The long-term objective of this research is to understand the complete vitamin K cycle in terms of structure and function. This series of reactions is important for post- translational modification of proteins involved in blood coagulation, bone formation, and a number of other physiological processes. The primary focus will be studying the structure and function of the enzyme, 3-glutamyl carboxylase, and its mechanism of action. The broader goal is to reproduce the carboxylation process, but in addition, the entire vitamin K cycle in vitro. This should help to understand the roles that carboxylase, vitamin K oxidoreductase, and any other essential components in the vitamin K cycle play in physiologic settings. Primarily, these studies will be designed to address the following: (1) determine the ways carboxylase recognizes and interacts with substrates and ligands;(2) identify and characterize catalytic residues crucial for 3-carboxylation;(3) elucidate the role of regulatory sites on the mechanism of 3-carboxylation;and (4) determine the three-dimensional structure of the 3-glutamyl carboxylase. In particular, the specific aims are as follows: Specific Aim 1: Investigate the role of substrate affinity in the control of vitamin K-dependent protein carboxylation in mammalian cells. Specific Aim 2: Identify residues important for substrate binding, propeptide binding, and the residues that mediate the allosteric interaction between these two sites;develop models compatible with our results to explain how carboxylase functions. Specific Aim 3: Identify the active site catalytic residues of carboxylase. Specific Aim 4: Investigate the three-dimensional structure of 3-glutamyl carboxylase. The pursuit of these goals will include creation of mutant carboxylases and biochemical characterization of these modified enzymes;use of cell culture and molecular biology techniques to investigate expression of pro-coagulation factors, with different affinities for carboxylase to study how carboxylation is controlled in a cellular milieu;and use electron crystallography to gain structural information that we will correlate with information from our functional studies to achieve a more complete understanding of the 3-glutamyl carboxylase.